The terbium ion is frequently used as a probe for calcium ion binding sites; it has a similar ionic radius but it binds with a higher affinity, and its fluorescent properties can provide additional information on bound ion coordination. Proteins involved in calcium ion regulated processes often contain a supersecondary structure called an EF-hand. Ligands for ion binding are contributed by six different amino acids in a single contiguous stretch of twelve amino acids. Calcium binding dissociation constants are typically sub-micromolar in such proteins. When the same amino acid sequence is synthesized as an isolated polypeptide, the calcium ion binding affinity becomes one hundred to one thousand times weaker. We have synthesized four such peptides, three of which contain disulfide crosslinks between residues that should be suitably oriented when an ion is bound to the polypeptide. The identification of these residues was based the several structures of EF-hand proteins available from X-ray crystallography. Experimental peptides contain either no disulfide, or disulfides connecting residues -1 to 13, 8 to 13 or -1 to 8, where residue 1 is the first amino acid in the sequence that provides a ligand to coordinate the ion. The goal of this research is to measure the extent that binding affinity is affected by the conformational constraints of the disulfide bonds in this relatively simple system. ITC has been employed because it provides a measurement of free energy of binding, but also the enthalpic and entropic contributions to binding. This information will be used in modeling the effect of the various modifications to the control peptide.